The advancement of our understanding of molecular genetics has lead to possibilities of treating diseases with gene-based therapies. Two major technical issues for efficient application of gene therapies is the delivery of the gene into the target cells and efficient, long-term expression of the gene. Likely the best strategy to achieve long-term expression of introduced gene will be to integrate introduced DNA into the genome of cell. The present inventors have developed a method which will increase significantly the efficiency of integration of introduced DNA into the genome of differentiated neurons.
The present inventors developed and tested a method which is based on the use of proteins involved in stimulation of the DNA repair and recombination process to integrate introduced DNA into adult neurons. DNA repair is carried out by DNA polymerases xcex4 (pol xcex4) and xcex5 (pol xcex5). These polymerases are stimulated by proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and replication factor C (RFC) under various conditions (Hunting et al., Biochemistry and Cell Biology, 69:303-310 (1991), Tsurimoto and Stillman, J. Biol. Chem., 266:1950-1960 (1991), Coverley et al., Nucleic Acids Research, 20:38730-3886 (1992), Melendy and Stillman, J. Biol. Chem., 268:3389-3395 (1993), Podust and Hubscher, Nucleic Acids Research, 22:2970-2983 (1993), Hubscher and Spadari, Physiological Rev., 74:259-285 (1994)). The same factors participate also in the process of DNA recombination. DNA damage caused by UV or gamma ray irradiation stimulates repair processes by inducing the expression of several repair proteins as well as activating enzymatic activities. Integration of foreign DNA occurs by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks (Murnane, et al., Nucl. Acids Res., 18:2733-2738 (1990)).
Levels of proteins that are involved in DNA repair are very low in the adult neurons. For example, expression of PCNA, RPA and RFC are almost undetectable in adult neurons. On the other hand, several components that are necessary for high efficiency recombination are expressed in neurons. For example, recombination activating gene RAG1 which is involved in the V(D)J gene recombination is expressed in many areas of the CNS (Chun et al., Cell, 64:189-200 (1991)). The present inventors propose that overexpression of proteins that are important components of repair and recombination system will result in increased integration of introduced DNA.
The homology between the DNA repair and recombination factors from different species (from yeast to human) is very high, which predicts that mechanisms operating in the regulation of these processes are similar in different species.
The present inventors have shown that transfecting neurons with DNA encoding PCNA and RFC large subunit in combination with UV treatment stimulates stable integration of introduced DNA into cortical neurons in vitro. The present inventors have also shown that such transfection in vitro can be applied to stably integrate a functional gene(s) by simultaneous cotransfection of the genes encoding PCNA and RFC with the functional gene(s).
Without being bound by any particular theory of how PCNA and RFC large subunit facilitate DNA integration, the present inventors hypothesize that by inducing DNA repair and recombination in differentiated neurons, the present invention provides for the stable production of specific proteins in neurons. With stable and functional integration of a therapeutic or functional gene, the present invention also provides for gene therapy in which normally differentiated neurons are induced to produce a given therapeutic protein.
The present invention is directed use of proteins involved in stimulation of DNA repair and recombination in neurons, which provides for production of proteins, peptides, or polypeptides (for simplicity, the term xe2x80x9cproteinxe2x80x9d is used to encompass all of these materials) in neurons, for gene therapy that provides proteins in neurons. According to the present invention, stable integration of delivered genes into the genome of neurons is accomplished. This novel method is based on the transfection of the postmitotic neurons with DNAs which are known to induce DNA repair and recombination, along with DNA which has potential therapeutic effect.
The present inventors demonstrated that introduced DNA expression was stable for weeks. With stable integration of the delivered gene into the genome of the target neurons, this method of gene transfer provides for gene therapy for neurodegenerative diseases such as Parkinson""s, Huntingtons and Alzheimers and for reconstruction following trauma and stroke. Further, this method can be used for gene transfer to any non proliferating and fully differentiated cell of the body, since regulation of the DNA repair and recombination is not unique in the neuronal cells and, likely, induction of DNA repair and recombination will result in stable integration of cotransfected DNA.
According to certain embodiments, the invention involves transfection and expression of DNA repair and recombination factors (for example, PCNA and large subunit of RFC) that stimulate DNA repair and recombination in differentiated target cells, in combination with UV irradiation to induce DNA repair and recombination. There are many factors that stimulate DNA repair and recombination that may be used in the present invention.
Since both UV and gamma ray irradiation stimulate DNA repair and recombination processes then it will be possible to use X-ray irradiation instead of UV irradiation in the present invention. According to certain embodiments, dosages of X-ray irradiation for in vitro use, may include exposing cell culture to a cesium (137Cs) irradiator at doses of about 10 to about 900 rad (1 rad equals 0.01 Gy). Humans and animals may be X-ray irradiated using an X-ray generator and different dose levels from 0.2 Gy to 50 Gy. According to certain preferred embodiments, irradiation will be focused on the treated area. Human and animal dosages may be calculated on results of in vitro experiments and by in vivo experiments as well.
According to certain embodiments, X-rays or other UV irradiation may not be necessary. Moreover, other means of damaging DNA to induce DNA repair and reconstruction may be employed according to certain embodiments.
Foreign DNA encoding therapeutically relevant proteins may be cotransfected during the induced DNA repair and recombination phase, which accomplishes a stable and functional integration of genes into neurons of different organisms including humans. This method provides gene-based therapies of the central nervous system (CNS).
For example, for Parkinson""s disease, cDNA encoding tyrosine hydroxylase, which is key enzyme in the synthesis of L-DOPA and dopamine, can be introduced into cathecholamine producing neurons. For other neurodegenerative diseases and for stroke and trauma patients, genes expressing neurotrophic factors and their receptors can be introduced to promote regeneration.
The following example illustrates the invention. This example is for illustrative purposes only and is not intended to limit the scope of the invention.